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Title: Investigating the mechanism of bone marrow failure observed in patients with acute myeloid leukaemia
Author: Hodby, Katharine Ailsa
ISNI:       0000 0004 7653 6549
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2018
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Patients with Acute Myeloid Leukaemia (AML) present with the signs and symptoms of bone marrow failure. This finding spans the genetic and phenotypic diversity of the disease. The mechanism which underlies it is poorly understood. This thesis explores the effect of AML on the normal haematopoietic stem cell (HSC) population, using primary human diagnostic bone marrow samples. Previous work from our group suggested that AML induces a state of quiescence in HSCs, producing a differentiation block responsible for the observed cytopenias1. Reversal of this process might offer an alternative to the current treatment of patients with palliative transfusions. I have developed a flow cytometry-based technique to differentiate normal HSCs from leukaemia cells, selecting cells with the CD34+38-ALDHhighCLL1- expression signature. Validation of this technique by assessment of sorted cells by FISH and PCR, suggests it is successful in 73% of AML samples. In a further 25% of samples, it selects for a population significantly enriched for normal HSCs. We used this panel to investigate the concentration of HSCs at AML diagnosis, compared to controls. We show that there is no significant difference between HSC concentration at AML diagnosis (n=38, median [HSC] 2.5 cells/μl) and controls (n=24, median [HSC] 2.4 cells/μl). HSC concentration was not significantly affected by AML karyotype, patient age or gender. However, those patients presenting with a low HSC concentration at diagnosis (< 0.1 HSC/μl) were found to have a significantly worse outcome both in terms of overall and relapse-free survival, an effect apparently independent of age, gender and underlying karyotype. HSC concentration at diagnosis with AML may therefore represent a new independent prognostic marker. We then studied CD33 expression patterns on HSCs within Core Binding Factor mutated AML (n=37) at diagnosis, and found its expression to be significantly lower than on HSCs within controls (n=9) (17% versus 58%, p=0.005). CD33 expression on HSCs from AML samples rose significantly from diagnosis to remission (n=16) (17% to 58%, p=0.0001). This mirrors previous findings from our group using CD34low AML samples, and is, we believe, the first time that the antigenic signature of normal HSCs has been shown to be modified. 6 by the presence of AML. However, an in vitro assay to test the significance of these changes in terms of the cytotoxicity of GO towards normal HSCs did not demonstrate a significant difference between HSC subgroups. Finally, we attempted to investigate the mechanism by which AML might induce HSC quiescence by studying the comparative transcriptomes of HSCs from CD34low AML (n=6) and controls (n=6) by RNA-Seq, using direct cell to cDNA synthesis, followed by amplification. A first attempt resulted in poor quality data, with a significant proportion of reads mapping to non-coding DNA regions. A repeat approach, using utilising immediate RNA extraction post sorting resulted in significantly better quality data Bioinformatics analysis revealed differential expression of 6 genes between the 2 datasets (GNPDA1, ADGRG3, MIAT, WDR31, RP11-244H3.1 and RXFP1). GO enrichment studies using David highlighted a number of pathways including the TNF signalling pathway (p=0.003; after Benjamini-Hochberg correction p=0.51). Validation of these findings by independent qPCR, and functional exploration of enriched signalling pathways remains outstanding.
Supervisor: Not available Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Acute Myeloid Leukaemia ; haematopoietic stem cells